Recombinant hirudin (r-hirudin) is being used increasingly for therapeutic anticoagulation in patients with heparin-induced thrombocytopenia undergoing cardiovascular surgery. Although multiple laboratory methods are available for measuring r-hirudin, the ecarin clotting time (ECT) is the most commonly used for this purpose. Ecarin (extracted from snake venom) converts prothrombin to meizothrombin, which promotes clot formation. Direct thrombin inhibitors, like r-hirudin, bind meizothrombin and yield a linear, dose-dependent prolongation of ECT. Low levels of prothrombin and fibrinogen in plasma samples can lead to higher ECT; suggesting falsely elevated r-hirudin levels. A modified ECT assay with prothrombin and fibrinogen in excess was optimized using an orthogonal array method to eliminate the variations in patients' plasma prothrombin and/or fibrinogen levels for accurate determinations of plasma r-hirudin levels. By using the modified ECT assay, falsely elevated r-hirudin levels can be avoided in patients undergoing cardiopulmonary bypass, thus providing reliable and accurate r-hirudin monitoring in this clinical setting.